为获得具有生物活性作用的猪乳铁蛋白肤LFcinP基因在巴斯德毕赤酵母中进行组成型表达,检索NCBI数据库公布的牛、羊、猪、骆驼等动物的乳铁蛋白及其抗菌肽的氨基酸序列,运用ExPASY在线网站,对猪乳铁蛋白保守区域所编码的蛋白进行理化性质分析,参照巴斯德毕赤酵母使用密码子偏好性,优化设计合成138bp编码猪乳铁蛋白肤LFcinP基因,通过pGAPHαM酵母组成型分泌表达载体将其整合到巴斯德毕赤酵母GS115菌株,获得表达猪乳铁蛋白肽LFcinP基因重组酵母菌株GS115p/GAPHαM-LFcinP.SDS-PAGE和Westemblot分析表明,获得5.4ku乳铁蛋白肽；分析96h的蛋白电泳带结果表明,重组蛋白占酵母菌上清蛋白35.9％,表达量为48.5μg·mL-1;动态生长曲线法抑菌试验结果表明,重组猪乳铁蛋白肽具有一定的抑菌作用.%To get consecutive expression of porcine lactoferricin gene (LFcinP) in Pichia pastorsis and detection of its antimicrobial activity, we calculated the gene of LFcinP on the basis of NCBI database about amino acid sequence form cattle, goat, porcine, camel and the relate documents' information, analyzed the sequence and physical and chemical properties; according to the amino acid sequences of porcine lactoferrin and the frequency of codon usage of Pichia pastorsis, the LFcinP gene was designed and synthesized. The LFcinP gene was cloned into the pGAPHaM vector to construct the recombinant plasmid pGAPHaM-LFcinP, the linearized plasmid was transformed into Pichia pastors GS115 strain by electroporation, and then got the recombinant strain GS115/pGAPHaM-LFcinP. The recombinant LFcinP protein with a molecular weight of 5.4ku was expressed in the supernatant identified by SDS-PAGE and Westernblot, and the SDS-PAGE results of 96h indicated that recombinant protein accounted for 35.9% of the total cellular protein, the concentration of expressed protein was about 48.5 μg·mL-1; the growth curve showed that the LFcinP had certain antibacterial abilities. It indicated that we had got the recombinant Pichia pastorsis consecutive expressing LFcinP, and this study provides a basis for the lactoferricin applied in the animal production.
展开▼
